Vaccines have been very effective in protecting people from a wide variety of diseases, whether caused by virus, bacteria, or fungus. The ability of vaccines to induce specific protection against such a wide range of pathogenic organisms results from their ability to stimulate specific humoral antibody responses, as well as cell-mediated responses. This invention relates to a process for preparing such vaccines, and particularly to a process for making protein-polysaccharide conjugates that are used in preparing vaccines, immunogens, and other valuable immunological reagents. The invention further relates to the vaccines, immunogens, and immunological reagents produced from the conjugates made according to the invention.
Certain agents can stimulate an immune response with minimal chemical modifications, for example, tetanus toxoid, which is immunogenic even in the absence of an adjuvant. Other important agents are either non-immunogenic or poorly immunogenic, but they can be converted into immunogenic molecules or constructs, in which form they can induce vigorous immune responses. For example, most polysaccharides are poorly immunogenic. After they are coupled to proteins, however, the resulting construct becomes immunogenic. The conjugation of proteins to polysaccharides converts the polysaccharide from a weakly immunogenic T-cell independent antigen to a T-cell dependent antigen that recruits T-cell help, and thus stimulates heightened immune responses. Note the discussion by J. M. Cruse, et al. (Editors), Conjugate Vaccines, Karger, Basel, (1989); and R. W. Ellis, et al. (Editors), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994). These books are entirely incorporated herein by reference.
Conjugation of a protein and a polysaccharide can provide other advantageous results. For example, it has been found that protein-polysaccharide conjugates enhance the antibody response not only to the polysaccharide component, but also to the protein component. This effect is described, for example, in the dual conjugate patent application of Mond and Lees, U.S. patent application Ser. No. 08/402,565 (filed Mar. 13, 1995); application Ser. No. 08/444,727 (filed May 19, 1995); and application Ser. No. 08/468,060 (filed Jun. 6, 1995). These patent applications each are entirely incorporated herein by reference. This effect also is described in A. Lees, et al., xe2x80x9cEnhanced Immunogenicity of Protein-Dextran Conjugates: I. Rapid Stimulation of Enhanced Antibody Responses to Poorly Immunogenic Molecules,xe2x80x9d Vaccine, Vol. 12, No. 13, (1994), pp. 1160-1166. This article is entirely incorporated herein by reference.
Noting at least some of the advantageous results obtained using protein-polysaccharide conjugates, researchers have developed various techniques to facilitate coupling of proteins and polysaccharides. Note W. E. Dick, et al., xe2x80x9cGlyconjugates of Bacterial Carbohydrate Antigens: A Survey and Consideration of Design and Preparation Factors,xe2x80x9d Conjugate Vaccines (Eds. Cruse, et al.), Karger, Basel, 1989, beginning at page 48. This article also is entirely incorporated herein by reference. As one example of a protein-polysaccharide coupling technique, the use of organic cyanylating reagents, such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate, also called xe2x80x9cCDAPxe2x80x9d in this patent application, has been developed. These reagents activate polysaccharides and facilitate coupling of polysaccharides to proteins for conjugate vaccines. The activated polysaccharides can be directly or indirectly coupled to proteins. The use of CDAP and other organic cyanylating reagents is described in the following U.S. Patent and Patent Applications of Andrew Lees: U.S. patent application Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned), U.S. Pat. No. 5,651,971; and U.S. patent application Ser. No. 08/482,666 (filed Jun. 7, 1995). This U.S. patent and the patent applications each are entirely incorporated herein by reference. The use of CDAP also is described in Lees, et al., xe2x80x9cActivation of Soluble Polysaccharides with 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate For Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents,xe2x80x9d Vaccine, Vol. 14, No. 3 (1996), pp. 190-198. This article also is entirely incorporated herein by reference. Other techniques for coupling proteins and polysaccharides using homobifunctional or heterobifunctional vinylsulfones are described in U.S. Provisional Patent Appln. No. 60/017,103 filed on May 9, 1996, and U.S. patent application Ser. No. 08/852,733 filed on May 7, 1997, each in the name of Andrew Lees. Protein/polysaccharide coupling using uronium salts and haloacyl reagents is described in U.S. Provisional Patent Appln. Nos. 60/041,781 (filed Mar. 24, 1997) and No. 60/042,379 (filed Apr. 24, 1997). These patent applications also are entirely incorporated herein by reference.
In the production of protein-polysaccharide conjugate vaccines, a major cost and time consuming step lies in the separation of the free protein (i.e., the unreacted or non-conjugated protein) from the conjugated protein-polysaccharide product. This separation, which is also called xe2x80x9cfractionation,xe2x80x9d usually is accomplished using a column chromatographic technique (e.g., size exclusion chromatography) or an ultrafiltration process. These protein separation processes significantly increase the time and expense involved in producing protein-polysaccharide conjugate vaccines. Under the good manufacturing procedure (xe2x80x9cGMPxe2x80x9d) guidelines, a dedicated (and expensive) chromatography column normally is needed for each type of vaccine conjugate to prevent contamination of the product.
In addition to the increased cost and time, this protein separation step often results in a significant loss of the desired protein-polysaccharide conjugate material. Additionally, the free protein material that is collected in this separation step typically is discarded. These factors further increase the costs involved in preparing a protein-polysaccharide conjugate vaccine.
It is an object of this invention to provide a method for producing protein-polysaccharide conjugates that avoids the problems and disadvantages described above. These conjugates can be used as intermediate materials in the production of other conjugates, such as hapten-protein-polysaccharide conjugates. It is a further object of this invention to provide vaccines, immunogens, and other immunological reagents that are produced by this method.
In one embodiment, this invention relates to a process for preparing a protein-polysaccharide conjugate. This process includes reacting a protein with a polysaccharide to produce a mixture including a protein-polysaccharide conjugate and free protein. At least one unreacted reagent or low molecular weight component is removed from this mixture to provide a purified mixture containing the protein-polysaccharide conjugate and free protein.
In another embodiment of the invention, a hapten-protein-polysaccharide conjugate is prepared. In this process, a purified mixture including a protein-polysaccharide conjugate and free protein first is produced in the manner described above. Thereafter, a hapten (e.g., a peptide) is reacted with the purified mixture of the protein-polysaccharide conjugate and the free protein, thereby providing a conjugate mixture including a hapten-protein conjugate, hapten-protein-polysaccharide conjugate, and free hapten. This conjugate mixture can be treated further to remove the free hapten to thereby provide a purified conjugate mixture including the hapten-protein-polysaccharide conjugate and the hapten-protein conjugate.
As another alternative, a hapten-protein conjugate first can be produced. As noted above, the hapten can be, for example, a peptide. The excess free protein and/or free hapten optionally can be removed at this stage. Thereafter, this conjugate, present in excess, is reacted with a polysaccharide to form a hapten-protein-polysaccharide conjugate. It is not necessary to remove the excess hapten-protein conjugate from the resulting conjugate mixture. The conjugate mixture includes the hapten-protein conjugate and the hapten-protein-polysaccharide conjugate.
The invention further relates to the protein-polysaccharide conjugate and free protein mixture, as well as the hapten-protein-polysaccharide conjugate and hapten-protein conjugate mixture, made by the processes of the invention. In addition to vaccines, the conjugates according to this invention can be used as immunogens or immunological reagents.